PRODUCTION AND CHARACTERIZATION OF BIOSURFACTANT BY Pseudomonas fluorescens USING CASSAVA FLOUR WASTEWATER AS MEDIA | Suryanti | Indonesian Journal of Chemistry 21281 40367 1 PB

Indo. J. Chem., 2013, 13 (3), 229 - 235

229

PRODUCTION AND CHARACTERIZATION OF BIOSURFACTANT
BY Pseudomonas fluorescens USING CASSAVA FLOUR WASTEWATER AS MEDIA
Venty Suryanti*, Soerya Dewi Marliyana, Desi Suci Handayani, and Desi Ratnaningrum
Department of Chemistry, Faculty of Mathematic and Natural Sciences, Sebelas Maret University
Jl. Ir. Sutami 36A, Surakarta, Central Java 57126, Indonesia
Received September 7, 2013; Accepted October 31, 2013

ABSTRACT
Biosurfactant with efficient emulsification properties could be produced by Pseudomonas flourescens using
cassava flour wastewater (manipueira) as media. The ability of P. flourescens to produce biosurfactant could
suggest potential use in industrial and environmental applications. Media containing a mixture of natural manipueira
and nutrient broth with 48 h fermentation was the optimum condition for the biosurfactant production. Based on UVVis and FT-IR spectra, the biosurfactant was indicated as rhamnolipids containing hydroxyl, ester, carboxylic and
aliphatic carbon chain functional groups. Biosurfactant exhibited critical micelle concentration (CMC) value of 715
mg/L and reduced the surface tension of the water from 80 mN/m to 59 mN/m. The biosurfactant was able to
decrease the interfacial tension about 51-70% when benzyl chloride, palm oil and kerosene were used as waterimmiscible compounds. The biosurfactant was able to form stable emulsion until 30 days when paraffin, soybean oil,
lubricant oil and kerosene were used as water-immiscible compounds.
Keywords: biosurfactant; manipueira; Pseudomonas flourescens; rhamnolipids

ABSTRAK
Biosurfaktan dengan sifat emulsifikasi yang baik dapat diproduksi oleh P. fluorescens menggunakan limbah
industri tepung tapioka (manipueira) sebagai media. Kemampuan produksi biosurfaktan oleh P. fluorescens bisa
diaplikasikan di industri dan untuk penyelesaian masalah lingkungan. Penggunaan campuran media nutrient broth
dan manipueira tanpa perlakuan awal dengan lama fermentasi 48 jam merupakan kondisi optimum untuk produksi
biosurfaktan. Analisa spektrofotometer UV-Vis dan FT-IR menunjukkan bahwa biosurfaktan merupakan
rhamnolipida yang mempunyai gugus hidroksi, ester, karboksilat dan rantai alifatik. Biosurfaktan mempunyai nilai
konsentrasi kritis misel (KKM) 715 mg/L dan mampu menurunkan tegangan permukaan air dari 80 mN/m ke
59 mN/m. Biosurfaktan ini dapat menurunkan tegangan muka sebesar 51-70% untuk senyawa hidrokarbon benzil
klorida, minyak sawit dan kerosene. Biosurfaktan mampu membentuk emulsi yang stabil sampai 30 hari untuk
senyawa hidrokarbon parafin, minyak kedelai, minyak pelumas dan kerosene.
Kata Kunci: biosurfaktan; manipueira; Pseudomonas flourescens; rhamnolipida
INTRODUCTION
Biosurfactants are a group of secondary
metabolites with surface active properties and are
synthesized by a great variety micro-organism. These
metabolites are complex amphiphilic molecules whose
hydrophobic and polar domains depend on the carbon
substrate and bacterial strain. Biosurfactants have

several advantages over the chemical surfactants, such
as lower toxicity, higher biodegradability [1], better
environmental compatibility [2], higher foaming [3],
higher selectivity and specific activity at extreme
temperatures, pH and salinity [4], and the ability to be
synthesized from renewable feedstock [5].
In previous studies, we found that biosurfactants
could be produced by P. aeruginosa using soybean oil
* Corresponding author. Tel/Fax : +62-271-663375
Email address : venty_s@yahoo.com

Venty Suryanti et al.

as media [6]. The biosurfactant was identified as
rhamnolipids and had the CMC of 860 mg/L and
reduced the surface tension of the water from 72 mN/m
to 52 mN/m. The biosurfactant could be used as an
emulsifier to form emulsion between water and
hydrocarbon such as palm oil, benzene, premium or
toluene with various stability. The results indicated that

biosurfactant could be used as an emulsifying and
emulsion-stabilizing agent.
Although productions of biosurfactants by several
micro-organisms and their application have been
reported [7-35], these compounds have not yet been
employed extensively in industry because of their high
production cost. The uses of low cost raw materials
appear as a natural choice to process overall economy.
Biosurfactant can be produced by microbial

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Indo. J. Chem., 2013, 13 (3), 229 - 235

fermentation processes using inexpensive agro-based
substrates and waste materials, such as peat
hydrolysate [36], olive oil mill effluent [36], lactic whey
[37], soybean curd residue [38], potato process effluent
[39-40] and molasses [41]. Cassava flour wastewater
(manipueira) is an example of agroindustrial waste or by

products that have high contents of carbohydrates for
the use of substrate for biosurfactants production. This
paper describes the biosurfactant production by
P. flourescens using manipueira as substrates and the
characterization of emulsifying properties of the
biosurfactants produced.
EXPERIMENTAL SECTION
Materials
All chemical were used are analytical grade from
Merck. P. flourescens was obtained from Food and
Nutrition Culture Collection (FNCC) 007 Inter University
Centre of Food and Nutrition Universitas Gadjah Mada,
Indonesia.
Instrumentation
Optical density was measured by Shimadzu UV160 1PC spectrometer and infrared spectra were
obtained by a Shimadzu FTIR-8201 PC spectrometer.
Procedure
Growth optimization conditions
Natural manipueira (NM), with the presence of
insoluble solids, was simply thawed, homogenized and

distributed in conical flasks. Decanted manipueira (DM),
with the absence of insoluble solids, was prepared by
heating the thawed waste until boiling to facilitate solids
removing. After cooling, the substrate was centrifuged
for 20 min and the supernatant was distributed in conical
flasks.
Experiments
on
growth
optimization
and
biosurfactant production were performed using five
-1
different media which all supplemented with 5 g.L NaCl.
-1
There were: NB, containing 8 g.L nutrient broth; M,
-1
containing NM; NBM containing NM and 8 g.L nutrient
broth; MS containing DM; NBMS containing DM and
-1

8 g.L nutrient broth. The pH of all media was adjusted
to 7.0.
The cultures were incubated at room temperature
on reciprocal rotary shaker (100 rpm at 30 °C) and were
monitored through bacterial growth, surface tension and
emulsification index (E24) for 12 days. Bacterial growth
was monitored by measuring the optical density at
365 nm by Shimadzu UV-160 1PC spectrometer.

Venty Suryanti et al.

Production medium and culture conditions
The bacterial isolates were streaked on a nutrient
agar slant and incubated for 24 h at 30 °C. A loop of
culture was inoculated in 5 mL of NB in a test tube and
incubated in a rotary shaker at 100 rpm at 30 °C for
21 h. An aliquot of 5 mL of inoculum was transferred to
250 mL of NBM media in a flask of 500 mL and
incubated in a rotary shaker of 100 rpm at 30 °C for
48 h.

Crude biosurfactant purification
For purification of biosurfactant, the pH of the
cultures supernatant fluid, obtained after removal of
cells by centrifugation for 10 min, was adjusted to pH
2.0 using 6 N HCl and allowed to stand overnight at
4 °C. This was followed by extraction with a mixture of
CHCl3 and CH3OH (2:1 v/v). The biosurfactant was
dried by NaSO4 anhydrous and evaporated on a rotary
evaporator [42-43].
Emulsification index (E24)
E24 of culture samples was determined by adding
2 mL of hydrocarbons and the same amount of
culture/distilled water, mixed by a vortex for 2 min, then
leaved to stand for 24 h. The E24 is given as
percentage of height of emulsified layer (mm) divided
by total height of the liquid column (mm) [44].
Surface tensions, interfacial tensions and CMC
The surface tension and interfacial tensions of the
biosurfactant were measured by the capillary rise
method at room temperature [45]. The purified

biosurfactant was dissolved in distilled water, and the
surface tension of the water was measured with
various concentrations of the biosurfactant. The
concentration at which micelles began to form was
represented as the CMC. At the CMC, sudden change
in the surface tension was observed. The CMC was
determined by plotting the surface tension as a function
of biosurfactant concentration.
Detection of rhamnolipids
A modified orcinol method was used for analyzing the
rhamnolipids using rhamnose as the standard [46]. A
culture supernatant (3.3 mL) was extracted twice with
diethyl ether (10 mL). The ether fractions were
evaporated to dryness and 0.5 mL of water was added.
After heating for 30 min at 80 °C the samples were
cooled at room temperature for 15 min and the optical
density readings by UV-Vis Spectrophotometer were
taken at 421 nm.
IR spectrometric analyses
Infrared (IR) absorption spectra were obtained by

a Shimadzu FTIR-8201 PC spectrometer in the range

Indo. J. Chem., 2013, 13 (3), 229 - 235

of 4000 to 400 cm
-1
1 cm .

-1

231

spectral region at a resolution of

Emulsification
properties
of
the
purified
biosurfactant

The interfacial surface tension and E24 of the
purified biosurfactant using different hydrocarbons (pen
tane, hexane, benzene, anisaldehyde, benzaldehyde,
toluene, benzyl chloride, aniline, paraffin, diesel, palm
oil, soybean oil, olive oil, lubricant oil and kerosene)
were tested. The purified biosurfactant (0.1 mg) was
added to a crew-capped tube containing distilled water
(1 mL) and the desired hydrocarbon (1 mL). The
interfacial tensions of the emulsions with and without
addition of purified biosurfactant were measured by the
capillary rise method at room temperature. The E24 of
the formed emulsions was monitored for 30 days.
Assay of emulsification activity and stability
Purified biosurfactant (3.3 mg) was diluted with
4 mL distilled water and 1 mL of hydrocarbons was
added. The mixture was shaken vigorously in a vortex
mixer for 2 min. The emulsion was allowed to sit for
10 min at room temperature, after which its absorbance
was measured at 540 nm. The absorbance was used to
express the emulsification activity. One unit of

emulsification activity was defined as that amount of
emulsifier that affected an emulsion with an absorbance
at 540 nm of 1.0.
The emulsion stability was analyzed based on the
emulsification activity [47]. The emulsified solutions were
allowed to stand for 10 min at room temperature and
then absorbance readings were also taken every 10 min
for 60 min. The log of the absorbance was then plotted
versus time. The slope (decay constant, Kd) of the line
was calculated, then expressed as emulsion stability.
RESULT AND DISCUSSION
Optimization of Biosurfactants Production
Experiments
on
growth
optimization
and
biosurfactant production were performed using five
different media: NB, M, NBM, MS and NBMS as
described in experimental sections. The experiment was
monitored through optical density, surface tension and
E24 for 12 days. Fig. 1 shows the time course of
biosurfactant production from P. fluorescens during
growth on manipueira as substrate. The surface tension
reached a minimum of 60.5 mN/m and the E24 reached
a maximal of 58% when the bacterial growth was at late
log phase which was about 2 days (48 h) after
inoculation (Fig. 1b and 1c). The bacterial growth was
continued to its stationary phase till 5 days (60 h) of
inoculation (Fig. 1a).

Venty Suryanti et al.

Fig 1. Growth of P. fluorescens in various media: (a)
optical density, (b) surface tension and (c).
emulsification index
The P. fluorescens were submitted to
biosurfactant production in two different manipueira
media: natural manipueira and decanted manipueira.
The purpose of this experiment was to evaluate the
viability of the use of manipueira waste in its natural
form what could reduce costs and facilitate medium
preparation. The condition that had the highest
microbial growth, surface tension reduction and E24 is
the best condition of biosurfactant production. Duncan
statistics analysis based on data shown on Fig. 1
shows that bacterial growth using natural manipueira
with the presence of nutrient broth as media with
2 days (48 h) of the fermentation time was the optimum
condition for the biosurfactant production.
As comparisons, biosurfactant production by P.
aeruginosa RB 28 (expressed as rhamnose
equivalents) reached the maximum level at the
stationary phase after 60 h of inuculation [48].
Production of biosurfactant from P. aeruginosa 44TI
started after 14 h of inoculation and reached its

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Indo. J. Chem., 2013, 13 (3), 229 - 235

maximal level after 58 h [49]. Rhamnolipid production
from P. aeruginosa GS3 started by 12 h and reached its
maximum at 72 h [50]. A reduction in the surface tension
of media as a result of biosurfactant production and
accumulation during the period between the expontial
growth and stationary phases has already been reported
for several other microorganisms. Since biosurfactants
are secondary metabolites, maximal glycolipid
production was found to accumulate at the stationary
stage of cell growth [51-52].
Detection of the Rhamnolipids
The rhamnose gave an absorption maximal at
417 nm. The UV-Vis spectra of the purified biosurfactant
also possessed an absorption maximal at 417 nm which
indicated that biosurfactant has rhamnose in its
structure. The FT-IR spectra of purified biosurfactant
showed the hydroxyl (-OH) stretching as a broad
-1
-1
absorption at 3400 cm . In the region 3000-2700 cm
were obtained several C-H stretching bands of CH2 and
CH3 groups. The deformation vibrations at 1458 and
-1
1381 cm also confirmed the presence of alkyl groups.
-1
Carbonyl stretching band was found at 1720 cm which
is characteristic for ester compounds. The ester carbonyl
-1
group was also proved from the band at 1095 cm which
corresponds to C-O deformation vibrations. FT-IR
spectra analyses of the biosurfactant were identical to
the rhamnolipids which was produced by P. fluorescens
using olive oil as a substrate and to the rhamnolipids
which was produced by P. putida using hexadecana or
glucose as a substrate [53]. FT-IR spectra analyses and
the result of modified orcinol method indicated that
P. fluorescens FNCC 007 produced a mixture of
rhamnolipids, the amphiphilic surface-active glycolipids
usually secreted by Pseudomonas spp.
CMC and the Surface Tension of the Biosurfactant
At the CMC, sudden changes in the surface
tension were observed. The CMC was determined by
plotting the surface tension as a function of biosurfactant
concentration. The CMC for the purified biosurfactant
was 715 mg/L. At the CMC, the purified biosurfactant
reduced the surface tension of the water from 80 mN/m
to 59 mN/m.
The CMC value of the biosurfactant is quite high
compared to those of other biosurfactants. For
examples, CMC value of rhamnolipids range from 27 to
40 mg/L [35-36] and that of trehalose lipid range from
4 to 15 mg/L [53-54]. At the CMC, the biosurfactant
reduced the surface tension of the water from 80 mN/m
to 59 mN/m. A good surfactant can lower surface tension
of water from 72 to 35 mN/m [42]. The surface tension for

Venty Suryanti et al.

Table 1. The interfacial surface tension of waterimmiscible compounds by biosurfactant.
Compounds
Pentane
Hexane
Benzene
Anisaldehyde
Benzaldehyde
Toluene
Aniline
Paraffin
Diesel
Palm oil
Soybean oil
Olive oil
Lubricant oil
Benzyl chloride
Kerosene

The decreased of interfacial
surface tension (%)
33
20
20
21
22
25
25
12
33
66
33
47
33
51
62

other biosurfactants also has been reported to range
from about 27 to 35 mN/m [55-57].
Capability of
Hydrocarbons

the

Biosurfactants

to

Emulsify

Addition of the biosurfactant decreased the
interfacial surface tension of the emulsions tested
(Table 1). About 51-70% decreased of interfacial
tension was showed when using benzyl chloride, palm
oil and kerosene as water-immiscible compounds;
31-50% decreased of interfacial tension was showed
when using pentane, diesel, olive oil, soybean oil and
lubricant oil as water-immiscible compounds; and
10-30% decreased of interfacial tension was showed
when
using
hexane,
benzene,
anisaldehyde,
benzaldehyde, toluene, aniline and paraffin as waterimmiscible compounds. The ability of the produced
biosurfactants to emulsify kerosene and crude oil is an
important feature for bioremediation applications.
On day one, more than 40% of emulsification
index were obtained when benzene, anisaldehyde,
toluene, palm oil, soybean oil used as water-immiscible
compounds (Table 2). It also showed that paraffin,
soybean oil, lubricant oil and kerosene formed stable
emulsion up to 30 days. The soybean oil, lubricant oil
and kerosene were then used as water-immiscible
compounds for emulsification activity and stability test.
As comparisons, biosurfactant produced from
P. aeruginosa RB 28 emulsified hydrocarbons,
hydrocarbons mixtures and vegetable oils and formed
stable emulsions. Emulsions formed with vegetable oils
are more stable [48]. It was reported that rhamnolipids
had the ability to emulsify efficiently hydrocarbons and
vegetable oils [43]. The stability of emulsions was
controlled and held stable for 21 days. The results
obtained with i-propyl palmitate (73%), castor oil (67%)

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Indo. J. Chem., 2013, 13 (3), 229 - 235

Table 2. The emulsification index of the water-immiscible compounds by biosurfactant.
Day
1
5
10
15
20
25
30

a
75
60
55
30
20
0
0

b
44
8
0
0
0
0
0

c
62
33
0
0
0
0
0

d
7
4
0
0
0
0
0

Emulsification index (%)
f
g
h
i
8
42
48
36
4
12
15
16
4
8
15
8
0
4
7
8
0
4
7
8
0
0
7
4
0
0
7
0

e
12
8
8
8
8
8
8

j
52
52
52
36
36
36
36

k
12
0
0
0
0
0
0

l
36
14
14
11
11
11
11

m
13
6
6
0
0
0
0

n
4
0
0
0
0
0
0

a = benzene; b = anisaldehyde; c = toluene; d = aniline; e = paraffin; f = diesel; g = palm oil; h = soybean oil;
i = olive oil; j = lubricant oil; k = benzyl chloride; l = kerosene; m = pentane; n = hexane

Table 3. Emulsification and stabilization properties of soybean oil, lubricant oil and kerosene by biosurfactant and
synthetic surfactants.
Decay constant
-3 b
(Kd, 10 )
-1.477
-9.211
-4.286

Soybean oil

Biosurfactant
Tween-80
Triton X-100

Emulsification activity
a
(OD540nm)
0.134
0.835
1.176

Lubricant oil

Biosurfactant
Tween-80
Triton X-100

0.380
1.986
2.430

-0.806
-1.170
-0.181

Kerosene

Biosurfactant
Tween-80
Triton X-100

0.482
2.060
0.717

-2.439
-0.789
-5.962

Compounds

Surfactants

or almond oil (83%) suggests a potential application in
the pharmaceutical and cosmetic industries [5].
Emulsifying
Activity
Biosurfactant

and

Stability

of

the

The emulsification activity and stability of the
biosurfactants and synthetic surfactants (Tween 80 and
Triton X-100) were examined with soybean oil, lubricant
oil and kerosene as water-immiscible substrates (Table
3). The stabilization ability of the biosurfactants was
described by the decay constant, Kd (the slope of the
emulsion decay plot) and the respective Kd values were
then calculated. The emulsification activity of the
biosurfactant was the lowest to that of the synthetic
surfactants tested, although the emulsion stability of the
biosurfactant was the greatest to that of the synthetic
surfactants tested when using soybean oil as waterimmiscible substrate. Emulsion stability of the purified
biosurfactant was among that of the synthetic
surfactants tested when using soybean oil or lubricant oil
as water-immiscible substrate.
The lower Kd shows the better stability of a
microemulsion. The biosurfactant exhibited good
emulsion stability indicating having high surface-active
properties. As a comparison, liposan from C. lipolytica
grown on YNB medium supplemented with 1%
hexadecane has emulsification activity of 0.75 and Kd of

Venty Suryanti et al.

-3

-6.0 x 10 for n-hexadecane as water-immiscible
compounds [48].
The ability of P. flourescens FNCC 007 to
produce biosurfactant using manipueira as media with
efficient emulsification properties, could suggest
potential use in industrial and environmental
applications.
CONCLUSION
Biosurfactant could be produced based on
microbial production by P. flourescens using cassava
flour wastewater (manipueira) as media. The optimum
condition for the biosurfactant production was obtained
using media containing a mixture of natural manipueira
and nutrient broth with 48 h fermentation. UV-Vis and
FT-IR spectra indicated that the biosurfactant was a
rhamnolipid containing hydroxyl, ester, carboxylic and
aliphatic carbon chain functional groups. Biosurfactant
exhibited CMC value of 715 mg/L. At the CMC, the
biosurfactant reduced the surface tension of the water
from 80 mN/m to 59 mN/m. The biosurfactant was able
to decrease the interfacial tension about 51-70% when
benzyl chloride, palm oil and kerosene were used as
water-immiscible compounds. The biosurfactant formed
stable emulsion until 30 days when paraffin, soybean
oil, lubricant oil and kerosene were used as waterimmiscible compounds.

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Indo. J. Chem., 2013, 13 (3), 229 - 235

ACKNOWLEDGEMENT
The financial support from the Directorate General
of National Higher Education, Department of National
Education, Indonesia in the form of Hibah Bersaing
Programs 2008 and 2009 with contract number of
017/SP2H/PP/DP2M/III/2008 and 231/D3/PL/2009 dated
24 March 2009 are greatly appreciated.
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